A SnakeMake workflow for Bulk RNA-seq

Reads were mapped onto genome with STAR, and adapters were removed with fastp.

For nomalisztion, gtftools was used to calculate gene_length and bioninfokit was used to give TPM, FPKM and CPM results.

For quality control, PCA plot, dendrogram plot and heatmap were used to show differences among samples or groups.

PyDESeq2 was used to perform differential expression anlysis.

General settings

To configure this workflow, modify config/config.yaml according to your needs, following the explanations provided in the file.

Sample sheet

• Add samples to config/samples.tsv. Only the column Sample is mandatory, but any additional columns can be added.
• For each sample, add one or more sequencing units (runs, lanes or replicates) to the Unit column of config/samples.tsv.
• For each sample, define Group column(experimental or clinical attribute).

To use

python run_rna.py \
    --fastqs fastq \
    --species mtu \
    --is_pair_end True \
    --samples sample.tsv \
    --outdir ./ \
    --compare compare.tsv \
    --pvalue 0.05 \
    --use_adjust_p True \
    --lfc 0.585 \
    --n_jobs 8

help

run_rna.py -h
usage: run_rna.py [-h] -f FASTQS -s {hsa,mmu,mtu} -p {True,False} -m SAMPLES -o OUTDIR [-c COMPARE] [-v PVALUE] [-u USE_ADJUST_P] [-l LFC] [-n N_JOBS] [-r] [-d]

RNA-seq

options:
  -h, --help            show this help message and exit
  -f FASTQS, --fastqs FASTQS
                        dir of fastq files
  -s {hsa,mmu,mtu}, --species {hsa,mmu,mtu}
                        species ABBR. one of hsa, mmu, mtu
  -p {True,False}, --is_pair_end {True,False}
                        out dir path
  -m SAMPLES, --samples SAMPLES
                        sample.tsv file path
  -o OUTDIR, --outdir OUTDIR
                        out_dir
  -c COMPARE, --compare COMPARE
                        compare.tsv file path
  -v PVALUE, --pvalue PVALUE
                        pvalue cutoff
  -u USE_ADJUST_P, --use_adjust_p USE_ADJUST_P
                        use adjust pvalue or not
  -l LFC, --lfc LFC     log2FoldChange cutoff
  -n N_JOBS, --n_jobs N_JOBS
                        no. of cpu to use
  -r, --rerun_incomplete
                        rerun incomplete pipline
  -d, --dry_run         fake run

Report

QC plot

Differential expression anlysis

MA plot

Volcano plot